Development of a microextraction by packed sorbent with gas chromatography-mass spectrometry method for quantification of nitroexplosives in aqueous and fluidic biological samples.

A new method for quantification of 12 nitroaromatic compounds including 2,4,6-trinitrotoluene, its metabolites and 2,4,6-trinitrophenyl-N-methylnitramine with microextraction by packed sorbent followed by gas chromatography and mass spectrometric detection in environmental and biological samples is developed. The microextraction device employs 4 mg of C18 silica sorbent inserted into a microvolume syringe for sample preparation. Several parameters capable of influencing the microextraction procedure, namely, number of extraction cycles, washing solvent, volume of washing solvent, elution solvent, volume of eluting solvent and pH of matrix, were optimized. The developed method produced satisfactory results with excellent values of coefficient of determination (R2  > 0.9804) within the established calibration range. The extraction yields were satisfactory for all analytes (> 89.32%) for aqueous samples and (> 87.45%) for fluidic biological samples. The limits of detection values lie in the range 14-828 pg/mL.

Biomonitoring of workers cleaning up ammunition waste sites.

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. In TNT-exposed humans, notable toxic manifestations have included aplastic anaemia, toxic hepatitis, cataracts, hepatomegaly and liver cancer. Therefore, it is important to develop protection measures and to monitor workers involved in the clean-up of ammunition sites. Haemoglobin (Hb) adducts of TNT, 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT), and the urine metabolites of TNT, 4ADNT and 2ADNT were found in 22-50% of the exposed workers, but not in the control group. The exposed workers were wearing protective equipment. The levels of erythrocytes, haemoglobin, creatinine, serum glutamic pyruvic transaminase and lymphocyte levels were significantly lower in the exposed workers than in the non-exposed workers. The levels of blood urea and reticulocytes were significantly higher in the exposed workers than in the non-exposed workers. Headache (26%), mucous membrane irritation (16%), sick leave (18%), lassitude (8%), anxiety (6%), shortness of breath (3%), nausea (5%) and allergic reactions (8%) were reported by the exposed workers. In a further analysis the U-4ADNT levels and the Hb-adduct levels were compared to the blood parameter and the health effects. The blood parameters were not significantly different between the U-4ADNT positive and U-4ADNT-negative group. Headache, mucous membrane irritation, sick leave, lassitude, anxiety, shortness of breath and allergic reactions were statistically not different between the two groups. Also in the workers with Hb-4ADNT adducts no significant negative changes were seen in regards to the changes of the blood parameters or the health effects. According to the results of the present study, it appears that the blood parameter changes and the health effects are more influenced by other factors than by the internal exposure to TNT.

Comparison of biomarkers in workers exposed to 2,4,6-trinitrotoluene.

2,4,6-Trinitrotoluene (TNT) is an important occupational and environmental pollutant. In TNT-exposed humans, notable toxic manifestations have included aplastic anaemia, toxic hepatitis, cataracts, hepatomegaly, and liver cancer. Therefore, methods were developed to biomonitor workers exposed to TNT. The workers were employed in a typical ammunition factory in China. The external dose (air levels and skin exposure), the internal dose (urinary metabolites), the biologically effective dose (haemoglobin adducts, urinary mutagenicity), biological effects (chromosomal aberrations and health effects), and individual susceptibility (genotypes of xenobiotic-metabolizing enzymes) were determined. Haemoglobin-adducts of TNT, 4-amino-2,6-dinitrotoluene (4ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT), and the urinary metabolites of TNT, 4ADNT and 2ADNT, were found in all workers and in some controls. The levels of the haemoglobin-adducts or the urinary metabolites correlated weakly with the skin or air levels of TNT. The urinary mutagenicity determined in a subset of workers correlated strongly with the levels of 4ADNT and 2ADNT in urine. The haemoglobin-adducts correlated moderately with the urinary metabolites and with the urinary mutagenicity. The genotypes of glutathione S-transferases (GSTM1, GSTT1, GSTP1) and N-acetyltransferases (NAT1, NAT2) were determined. In general, the genotypes did not significantly influence the haemoglobin-adduct levels and the urine metabolite levels. However, TNT-exposed workers who carried the NAT1 rapid acetylator genotype showed an increase in urinary mutagenicity and chromosomal aberrations as compared with slow acetylators. The haemoglobin adduct 4ADNT was significantly associated with a risk of hepatomegaly, splenomegaly and cataract; urine metabolites and genotypes were not associated with health effects. These results indicate that a set of well-selected biomarkers may be more informative regarding exposure and effect than routinely performed chemical measurements of pollutants in the air or on the skin.

Fate and effects of 2,4,6-trinitrotoluene (TNT) from dumped ammunition in a field study with fish and invertebrates.

2,4,6-Trinitrotoluene (TNT) is the major explosive ingredient in ammunition dumped into lakes and sea after World War II. The aim of the present field study was to study the fate and effect of TNT and its degradation products from dumped ammunition. Artillery shells were cleaved longitudinally to expose TNT and placed in open boxes filled with sediment, and then placed at the sea bottom. Sediment samples were taken in each box at the start and after 3, 9, 13, 20, 24, 33, and 36 months, and the sediments were tested for toxicity with bioassays using Nitocra spinipes (96 h), Hyalella azteca (96 h), and Daphnia magna (24 and 48 h). The result from the bioassays showed no impact of dumped ammunition on the survival of H. azteca and mobility of D. magna. Bioassays with N. spinipes showed significant differences in toxicity between control boxes and boxes with shells after 9 months and thereafter. The mean mortality (+/- SD) of N. spinipes in boxes with shells was 63 +/- 22%, and the mortality in control boxes was 23 +/- 17%. No continuous increase in sediment toxicity over time was found. After 3 years, cages with European flounder (Platichtys flesus) and blue mussels (Mytilus edulis) were attached to the boxes. The fish were examined for biochemical and physiological effects 8 weeks later. Exposure to ammunition, which had rested on the sea bottom 3 years, caused no significant effects on body indices, hematological variables, and detoxification and antioxidant enzymes activities in the flounder. The sediment, bile, and blood plasma of exposed fish, and hepatopancreas of exposed mussels, contained no detectable levels of TNT and its metabolites. Only minor disappearance of TNT from the shells could be detected by visual inspection on site (by scuba divers). This study suggests that the survival of sensitive benthic organisms, e.g., N. spinipes, might be negatively affected at an ammunition dumping site.

Interleukin-6 and tumor necrosis factor alpha in relation to myocardial infarct size and collagen formation.

BACKGROUND: Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) levels increase after acute myocardial infarction (AMI) in humans. Experimental data suggest that these cytokines regulate the initiation of scar formation after AMI. We investigated the interrelationships of IL-6 and TNF-alpha, tissue injury, infarct size, cardiac function, and collagen formation in humans. METHODS: Serum and plasma samples were taken on 93 patients receiving thrombolytic treatment for their first AMI. Collagen formation was evaluated by measuring concentrations of serum aminoterminal propeptide of type III procollagen (PIIINP). RESULTS: IL-6 levels increased by 44% (P<.001) and peaked at 24 hours. Peak IL-6 levels correlated positively with area under the curve of creatine kinase MB mass (r=.31, P<.01), peak troponin T level (r=.34, P<.005), and PIIINP measured at discharge (r=.46, P<.001). There were no changes in TNF-alpha levels, and patients with left ventricular dysfunction (EF<40%) had similar TNF-alpha levels as those with preserved left ventricular function. CONCLUSIONS: IL-6 may regulate collagen formation and thus remodeling of the left ventricle after AMI. In addition, TNF-alpha measurement is useless in the assessment of infarct size or left ventricular function during the immediate post-infarction period.

Monitoring human risk and exposure to trinitrotoluene (TNT) using haemoglobin adducts as biomarkers.

Two studies were carried out in a trinitrotoluene (TNT) plant using TNT haemoglobin (TNT-Hb) adduct as a biomarker to study dose-adduct and adduct-response relationships. In the first study, TNT-Hb adduct levels were determined in 117 TNT-exposed workers in different working sites with different exposure conditions. External exposure was calculated from the inhaled air concentration plus skin contamination. TNT-Hb adduct levels in blood were significantly correlated with their external exposure to TNT. Two methods, HPLC-UV and CI-ELISA, were developed for measuring TNT-Hb adduct: good correlations (r = 0.77 and 0.86) were found between these 2 methods. In the second study, TNT cataract was used as an indicator of health effects. The prevalence of cataract and the degree of lenticular damage increased with the increase of blood TNT-Hb level.

In vivo covalent binding of [14C]trinitrotoluene to proteins in the rat.

When a single dose of [14C]trinitrotoluene was administered intraperitoneally (i.p.) to rats at 1, 10 or 50 mg/kg of body weight, covalently bound radioactivity was detected in globin, plasma proteins and proteins in the liver and kidney. The extent of covalent binding was dose dependent and was highest in plasma and renal proteins at all times up to 4 h after dosing. Covalent adduct levels in globin, however, decline slower than others. At a dose of 50 mg/kg of body weight, globin covalent adduct levels peaked at 1 h after dosing at 182 pmol/mg protein and subsequently decreased to approximately 50 pmol/mg protein between days 1 and 8. Of the covalent adduct levels in liver and kidney, those in the 10,000 x g and microsomal fractions were found to be higher than that in the cytosolic fraction. Radioactivity covalently bound to globin and the hepatic proteins was susceptible to dilute acid hydrolysis from which 2-amino-4,6-dinitrotoluene (2A) and 4-amino 2,6-dinitrotoluene (4A) were the major products recovered by solvent extraction. Upon acetylation, the hydrolysate gave rise to derivatives identified as the acetates of 2A and 4A on the basis of mass spectrometry and HPLC cochromatography with authentic samples. Four hours after an i.p. dose of [14C]TNT at 50 mg/kg of body weight about 0.4% of the dose was found as bound adducts to hemoglobin, of which approximately 48% was recovered as solvent extractable radioactivity after acid hydrolysis. About 2% of the radioactive dose was in the liver, of which approximately 30% was covalently bound to hepatic proteins, and approximately 49% of that was convertible to solvent extractable radioactivity upon acid hydrolysis. In vitro incubation of [14C]TNT with blood showed that there was a linear increase of covalent adducts in globin during the first 2 h of incubation; the concentration of covalent adducts was slightly higher than that with plasma proteins. The major compounds recovered from the hydrolysate of the globin adducts were also 2A and 4A as obtained from globin in the in vivo studies. On the basis of the in vitro and in vivo study results, we have confirmed the formation of protein adducts following a single i.p. administration of [14C]TNT at 1, 10 or 50 mg/kg of body weight to the rat or by in vitro incubation with blood.(ABSTRACT TRUNCATED AT 400 WORDS)

Applications of liquid chromatography-mass spectrometry in metabolic studies of explosives.

Liquid chromatography-mass spectrometry was used for the detection and identification of metabolites of 2,4,6-trinitrotoluene (TNT) in urine and blood. The metabolites were found in the urine of rats and in the blood of rabbits fed with TNT, in the urine of rats exposed to TNT by skin absorption and in the urine of TNT munition workers. The detected metabolites, formed by reduction processes, included 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2,6-diamino-4-nitrotoluene, in addition to untransformed TNT.